Kathryn H. Bradley

Increased cystine in leukocytes from individuals homozygous and heterozygous for cystinosis.

In patients with cystinosis, the concentration of free cystine in leukocytes was 80 times greater than normal, and six times the normal content for their parents. This is the first demonstration of an abnormality in heterozygotes for this rare inherited disease of childhood. Three-quarters of the cystine was recovered in the granular fraction of cystinotic leukocytes.

Increased free-cystine content of fibroblasts cultured from patients with cystinosis

The presence of a significantly increased content of free-cystine in skin fibroblasts from both homozygotes and heterozygotes for cystinosis emphasizes the central role of cystine in this disease, even though the primary defect responsible for cystine accumulation is yet to be determined. The studies described in this communication provide evidence that cystine is compartmentalized in a subcellular location in cystinotic cells. In fact, the very growth of cystinotic fibroblasts in the presence more than 100 times the usual content of free-cystine is evidence that the accumulated cystine is not freely dispersed throughout the cell, since would otherwise inhibit many enzymes requiring free sulfhydryl groups for activity (Patrick, 1965). We have no evidence as to whether the cystine is located in a known subcellular organelle or in a previously unrecognized location. Skin fibroblasts may provide a convenient tool to pursue these questions.

Cells, collagen and idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a usually fatal disorder of lung with clearly defined clinical, roentgenographic, physiologic, morphologic, scintigraphic and bronchoalveolar lavage features. Current concepts of the pathogenesis of this disorder suggest a central role for a chronic alveolitis in causing changes in parenchymal cell populations and derangements in interstitial collagen. Of the many inflammatory and immune effector cells comprising the alveolitis of IPF, it is likely that the neutrophil is the most important mediator of parenchymal damage. To follow the status of lung neutrophils in patients with this disease, two methods have been utilized. Both gallium-67 scanning and bronchoalveolar lavage quantitate the extent of the alveolitis and can be used to stage and follow these patients. The treatment of IPF remains controversial, but it is likely that corticosteroids reduce the alveolitis and prolong the lifespan of these patients.

Collagen in the human lung. Quantitation of rates of synthesis and partial characterization of composition.

The presence of collagen in lung is fundamental in normal lung structure and function. Methods have been developed to examine human fetal and adult lung collagen with respect to its composition and synthesis. The second trimester fetal lung has a large number of cells per unit lung mass (36.6 plus or minus 2.7 mug DNA/mg dry wt) and relatively small amounts of collagen (17.0 plus or minus 5.3 mug collagen/mg dry wt). The number of cells per unit lung mass in the adult lung (11.1 plus or minus 3.4 mug DNA/mg dry wt) is 30% of the number of cells in the fetal lung, but the adult has 11 times more collagen (196 plus or minus 25 mug collagen/mg dry wt). The composition of fetal lung collagen can be partially characterized by extraction with salt at neutral pH, acetic acid, or guanidine. The extracted chains, representing 10% of the total lung collagen, chromatograph as alpha1 and alpha2 chains, each with a mol wt of 100,000 and an animo acid composition characteristic for collagen but not specific for lung. Short-term explant cultures of fetal and adult lung synthesize alpha chains which can be isolated by ion-exchange chromatography. These chains, representing 30-40% of the total collagen synthesized by the explants, coelectrophorese with extracted collagen chains on acrylamide gels: they are destroyed by clostridial collagenase and they have a mol wt of 100,000. Although the composition of the collagen synthesized by these explants can be only partially characterized, the rate of synthesis of both collagen and noncollagen protein can be quantitated. In fetal lung, 4.0 plus or minus 1.2% of the amino acids incorporated into protein per hour are incorporated into collagen. In normal adult lung, this percentage (4.2 plus or minus 0.9%) is remarkably similar. These values are almost identical to the relative rate of collagen synthesis in rabbit lung in the same age range. This technology should be applicable to answer specific questions regarding collagen synthesis and degradation in human lung disease.

Cystine: Compartmentalization within Lysosomes in Cystinotic Leukocytes

The large amount of cystine compartmentalized in cystinotic leukocytes cosediments in isopycnic sucrose density gradients with dense lysosomal particles, within which it is presumably contained. Such cystine appears to be primarily noncrystalline in these organelles.

Lung collagen heterogeneity. Synthesis of type I and type III collagen by rabbit and human lung cells in culture.

The fetal and adult lung have a constant level of collagen synthesis that represents 4-5% of the total amino acids incorporated into lung protein. Prior studies have demonstrated that this collagen is not homogeneous but rather is composed of at least two collagen types, I and II, each localized to specific lung structures. Although it is known that explants of rabit lung parenchyma and blood vessels synthesize type I collagen and that rabbit lung tracheobronchial tree synthesizes type II collagen, it has been suggested that other collagen types are present in lung. It is not known which cells are responsible for the synthesis of any lung collagen type. To approach the problem of additional lung collagen heterogeneity and the identification of the cells responsible for lung collagen synthesis, techniques were developed to examine collagen synthesized by lung cells in culture. 10-15% of the proteins synthesized by confluent cultures of rabbit lung cells and fetal human lung fibroblasts are collagen. Separation and purification of this collagen by ion-exchange chromatography and cyanogen bromide (CNBr) peptide mapping techniques indicate that collagen secreted by these cells is composed of two collagen types, I and III. The CNBr peptides of type I collagen secreted by these cells are identical to the CNBr peptides of type I collagen synthesized by lung parenchyma and blood vessels. The peptides of type III collagen secreted by these cells are identical to fetal skin type III collagen CNBr peptides. The existence of 40 cell types and the insolubility of lung collagen increase the complexity of identifying the types of collagen in lung and the cells responsible for the synthesis of each type. The techniques described here should eventually lead to a complete description of the synthesis and composition of lung collagen, thus providing a probe to understand the role of collagen in lung development and structure in health and disease.

Chapter 3 The Fibroblast of Human Lung Alveolar Structures: A Differentiated Cell with a Major Role in Lung Structure and Function

Publisher Summary This chapter describes the importance of the fibroblast of the human lung-alveolar structures. The postnatal lung is a complex organ with function to mediate the exchange of oxygen and carbon dioxide between the atmosphere and the blood. The lung brings air and blood into close proximity in structures termed “alveoli.” In the adult human lung, alveoli are comprised of four major cell types. The fibroblast significantly influences alveolar structure and function through its major role in maintaining the interstitial connective tissue. Morphological studies have demonstrated that human fibroblasts are also capable of ingesting collagen fibrils and thus use phagocytotic mechanisms to modulate the connective tissue matrix. Lungs were obtained from human fetuses following abortion and stored briefly in a sterile culture medium until processing could be initiated. After becoming confluent, pulmonary fibroblasts in the tissue culture measure up to 100 μpm in length and 10 pm in maximal width. The chromatin is evenly distributed with only minimal margination.

Biochemical Comparisons of the Adult and Childhood Forms of Cystinosis

Abstract Three unrelated adult patients with cystinosis were studied in an attempt to explain the differences in the clinical manifestations of childhood and adult cystinosis. The free cystine cont...

Transport and Intracellular Fate of Cysteine-35S in Leukocytes from Normal Subjects and Patients with Cystinosis

Extract: The transport and intracellular fate of cysteine-35S was studied in leukocytes from nine children with cystinosis and nine control subjects of similar age in an attempt to explain the presence of 80 times the normal quantity of free cystine in cystinotic leukocytes. Two differences were observed: the cystinotic cells took up nearly twice as much cysteine-35S as did control cells. Subsequently, almost 40 percent of the 35S was found as the oxidized form, cystine-35S in cystinotic leukocytes, compared with only 2 percent in control leukocytes. Comparison of intracellular and extracellular concentrations of cysteine-35S at steady state showed no differences between normal and cystinotic leukocytes. Both cell types incorporated 30 percent of the intracellular 35S into glutathione and showed the same time course of 35S efflux.Speculation: The increased uptake of cysteine-35S shown by cystinotic leukocytes could be taken as valid conventional evidence for a primary defect in the transport of cysteine in cystinosis. The prompt appearance of the extra 35S transported as intracellular cystine-35S suggests, however, that this rapid conversion may be the primary defect and that the increased uptake of cysteine-35S is but a compensatory mechanism for maintaining the physiological concentration of intracellular cysteine.

Identification ofheterozygous genotype for cystinosis in utero by a new pulse-labeling technique: Preliminary report

U s l x o a new method developed for the study of small numbers of cystinotic cells in vitro, we have made a probable identification in utero of the heterozygous state of nephropathic cystinosis. The data imply that diagnosis of the homozygous state may also be possible early in gestation. Schneider and associates ~ demonstrated a 100-fold increase in content of free cystine in fibroMasts cultured from the skin of cystinotic children when compared to normal children (Table I ) , and a lesser increase of about sixfold in the fibroblasts of most individuals heterozygous for cystinosis. 1 In other heritable disorders, the expression of the biochemical abnormality in cultured fibroblasts has been accompanied by a similar expression in cells cultured from the amniotic fluid of an affected fetus, 2-* although one apparent exception to this rule has recently been found. ~ However, the large number of cells required for the usual chemical determination of cystine content